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1.
Maribel L. Dionisio-Sese Atsuhiro Shimada Tadashi Maruyama Shigetoh Miyachi 《Archives of microbiology》1993,159(1):1-5
The prokaryotic algal symbiont of ascidians, Prochloron sp., was found to exhibit carbonic anhydrase activity which is largely associated with the cell surface. This extracellular carbonic anhydrase activity was inhibited, while the intracellular activity was not affected, by chloride or bromide. Acetazolamide and ethoxyzolamide inhibited carbonic anhydrase activity with I50 values of 7×10-4 and 3×10-4M, respectively. These I50 values are similar to those observed for intracellular carbonic anhydrases of Synechococcus sp. PCC7942, Chlamydomonas reinhardii and spinach.Abbreviations AZA
acetazolamide
- CA
carbonic anhydrase
- chl
chlorophyll
- EZA
ethozyzolamide
- I50
concentration of an inhibitor required to cause 50% inhibition
- Rubisco
ribulose-1,5-bisphosphate carboxylase/oxygenase
- U
unit 相似文献
2.
The Role of External Carbonic Anhydrase in Inorganic Carbon Acquisition by Chlamydomonas reinhardii at Alkaline pH 总被引:9,自引:9,他引:0
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The role of external carbonic anhydrase in inorganic carbon acquisition and photosynthesis by Chlamydomonas reinhardii at alkaline pH (8.0) was studied. Acetazolamide (50 micromolar) completely inhibited external carbonic anhydrase (CA) activity as determined from isotopic disequilibrium experiments. Under these conditions, photosynthetic rates at low dissolved inorganic carbon (DIC) were far greater than could be maintained by CO2 supplied from the spontaneous dehydration of HCO3− thereby showing that C. reinhardii has the ability to utilize exogenous HCO3−. Acetazolamide increased the concentration of DIC required to half-saturate photosynthesis from 38 to 80 micromolar, while it did not affect the maximum photosynthetic rate. External CA activity was also removed from the cell-wall-less mutant (CW-15) by washing. This had no effect on the photosynthetic kinetics of the algae while the addition of acetazolamide to washed cells (CW-15) increased the K½DIC from 38 to 80 micromolar. Acetazolamide also caused a buildup of the inorganic carbon pool upon NaHCO3 addition, indicating that this compound partially inhibited internal CA activity. The effects of acetazolamide on the photosynthetic kinetics of C. reinhardii are likely due to the inhibition of internal rather than a consequence of the inhibition of external CA. Further analysis of the isotopic disequilibrium experiments at saturating concentration of DIC provided evidence consistent with active CO2 transport by C. reinhardii. The observation that C. reinhardii has the ability to take up both CO2 and bicarbonate throws into question the role of external CA in the accumulation of DIC in this alga. 相似文献
3.
Carbonic anhydrase III from rabbit muscle, a newly discovered major isoenzyme of carbonic anhydrase, has been found to be also a p-nitrophenyl phosphatase, an activity which is not associated with carbonic anhydrases I and II. The p-nitrophenyl phosphatase activity has been shown to chromatograph with the CO2 hydratase activity; both activities are associated with each of its sulfhydryl oxidation subforms; and both activities follow the same pattern of pH stability. This phosphomonoesterase activity of carbonic anhydrase III has an acidic pH optimum (<5.3); its true substrate appears to be the phosphomonoanion with a Km of 2.8 mm. It is competitively inhibited by the typical acid phosphatase inhibitors phosphate (Ki = 1.22 × 10?3M), arsenate (Ki = 1.17 × 10?3M), and molybdate (Ki = 1.34 × 10?7M), with these inhibitors having no effect on the CO2 hydratase or the p-nitrophenyl acetate esterase activities of carbonic anhydrase III. The p-nitrophenyl acetate esterase activity of carbonic anhydrase III, on the other hand, has the sigmoidal pH profile with an inflection at neutral pH, typical of carbonic anhydrases for all of their substrates, and is inhibitable by acetazolamide (a highly specific carbonic anhydrase inhibitor) to the same degree as the CO2 hydratase activity. The acid phosphatase-like activity of carbonic anhydrase III is slightly inhibited by acetazolamide at acidic pH, and inhibited to nearly the same degree at neutral pH. These data are taken to suggest that the phosphatase activity follows a mechanism different from that of the CO2 hydratase and p-nitrophenyl acetate esterase activities and that there is some overlap of the binding sites. 相似文献
4.
Effect of Carbonic Anhydrase Inhibitors on Inorganic Carbon Accumulation by Chlamydomonas reinhardtii 总被引:32,自引:17,他引:15
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Membrane-permeable and impermeable inhibitors of carbonic anhydrase have been used to assess the roles of extracellular and intracellular carbonic anhydrase on the inorganic carbon concentrating system in Chlamydomonas reinhardtii. Acetazolamide, ethoxzolamide, and a membrane-impermeable, dextran-bound sulfonamide were potent inhibitors of extracellular carbonic anhydrase measured with intact cells. At pH 5.1, where CO2 is the predominant species of inorganic carbon, both acetazolamide and the dextran-bound sulfonamide had no effect on the concentration of CO2 required for the half-maximal rate of photosynthetic O2 evolution (K0.5[CO2]) or inorganic carbon accumulation. However, a more permeable inhibitor, ethoxzolamide, inhibited CO2 fixation but increased the accumulation of inorganic carbon as compared with untreated cells. At pH 8, the K0.5(CO2) was increased from 0.6 micromolar to about 2 to 3 micromolar with both acetazolamide and the dextran-bound sulfonamide, but to a higher value of 60 micromolar with ethoxzolamide. These results are consistent with the hypothesis that CO2 is the species of inorganic carbon which crosses the plasmalemma and that extracellular carbonic anhydrase is required to replenish CO2 from HCO3− at high pH. These data also implicate a role for intracellular carbonic anhydrase in the inorganic carbon accumulating system, and indicate that both acetazolamide and the dextran-bound sulfonamide inhibit only the extracellular enzyme. It is suggested that HCO3− transport for internal accumulation might occur at the level of the chloroplast envelope. 相似文献
5.
Mass Spectrometric Measurement of Intracellular Carbonic Anhydrase Activity in High and Low C(i) Cells of Chlamydomonas: Studies Using O Exchange with C/O Labeled Bicarbonate
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By measuring 18O exchange from doubly labeled CO2 (13C18O18O), intracellular carbonic anhydrase activity was studied with protoplasts and chloroplasts isolated from Chlamydomonas reinhardtii grown either on air (low inorganic carbon [Ci]) or air enriched with 5% CO2 (high Ci). Intact low Ci protoplasts had a 10-fold higher carbonic anhydrase activity than did high Ci protoplasts. Application of dextran-bound inhibitor and quaternary ammonium sulfanilamide, both known as membrane impermeable inhibitors of carbonic anhydrase, had no influence on the catalysis of 18O exchange, indicating that cross-contamination with extracellular carbonic anhydrase was not responsible for the observed activity. This intracellular in vivo activity from protoplasts was inhibited by acetazolamide and ethoxyzolamide. Intracellular carbonic anhydrase activity was partly associated with intact chloroplasts isolated from high and low Ci cells, and the latter had a sixfold greater rate of catalysis. The presence of dextran-bound inhibitor had no effect on chloroplast-associated carbonic anhydrase, whereas 150 micromolar ethoxyzolamide caused a 61 to 67% inhibition of activity. These results indicate that chloroplastic carbonic anhydrase was located within the plastid and that it was relatively insensitive to ethoxyzolamide. Carbonic anhydrase activity in crude homogenates of protoplasts and chloroplasts was about six times higher in the low Ci than in high Ci preparations. Further separation into soluble and insoluble fractions together with inhibitor studies revealed that there are at least two different forms of intracellular carbonic anhydrase. One enzyme, which was rather insoluble and relatively insensitive to ethoxyzolamide, is likely an intrachloroplastic carbonic anhydrase. The second carbonic anhydrase, which was soluble and sensitive to ethoxyzolamide, is most probably located in an extrachloroplastic compartment. 相似文献
6.
《Developmental biology》1986,116(2):548-551
Red cell carbonic anhydrase activity, 2.3 DPG concentration, and activities of three key enzymes controlling DPG metabolism (PK, PFK and DPGM) were measured in normoxic and hypoxic (incubation in 13.5% O2) chick embroys. In normoxia 2.3 DPG concentration and carbonic anhydrase activity begin to increase by the third week of incubation. Hypoxia induces a rise of 2.3 DPG concentration and carbonic anhydrase activity by Day 8 of development, i.e., 1 week earlier. Since during normal development chick embryos become hypoxic in the third week of incubation, the results suggest that PO2 has a controlling influence on the timing of differentiation events of definitive embryonic red cells. 相似文献
7.
Carbonic anhydrase activities of pea thylakoids as well as thylakoid fragments enriched either in Photosystem 1 (PS1-membranes)
or Photosystem 2 (PS2-membranes) were studied. The activity of PS1-membranes if calculated on chlorophyll basis was much higher
than the activity of PS2-membranes. Acetazolamide, a non-permeable inhibitor of carbonic anhydrases, increased carbonic anhydrase
activity of PS2-membranes at concentrations lower than 10−6 M and suppressed this activity only at higher concentrations. A lipophilic inhibitor of carbonic anhydrases, ethoxyzolamide,
effectively suppressed the carbonic anhydrase activity of PS2-membranes (I
50 = 10−9 M). Carbonic anhydrase activity of PS1-membranes was suppressed alike by both inhibitors (I
50 = 10−6 M). In the course of the electrophoresis of PS2-membranes treated with n-dodecyl-β-maltoside “high-molecular-mass” carbonic anhydrase activity was revealed in the region corresponding to core-complex
of this photosystem. Besides, carbonic anhydrase activity in the region of low-molecular-mass proteins was discovered in the
course of such an electrophoresis of both PS2-and PS1-membranes. These low-molecular-mass carbonic anhydrases eluted from
corresponding gels differed in sensitivity to specific carbonic anhydrase inhibitors just the same as PS1-membranes versus
PS2-membranes. The results are considered as evidence for the presence in the thylakoid membranes of three carriers of carbonic
anhydrase activity.
Published in Russian in Biokhimiya, 2006, Vol. 71, No. 5, pp. 651–659. 相似文献
8.
Identification of Extracellular Carbonic Anhydrase of Chlamydomonas reinhardtii 总被引:20,自引:17,他引:3
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We have examined the induction of carbonic anhydrase activity in Chlamydomonas reinhardtii and have identified the polypeptide responsible for this activity. This polypeptide was not synthesized when the alga was grown photoautotrophically on 5% CO2, but its synthesis was induced under low concentrations of CO2 (air levels of CO2). In CW-15, a mutant of C. reinhardtii which lacks a cell wall, between 80 and 90% of the carbonic anhydrase activity of air-adapted cells was present in the growth medium. Furthermore, between 80 and 90% of the carbonic anhydrase is released if wild type cells are treated with autolysin, a hydrolytic enzyme responsible for cell wall degradation during mating of C. reinhardtii. These data extend the work of Kimpel, Togasaki, Miyachi (1983 Plant Cell Physiol 24: 255-259) and indicate that the bulk of the carbonic anhydrase is located either in the periplasmic space or is loosely bound to the algal cell wall. The polypeptide associated with carbonic anhydrase activity has a molecular weight of approximately 37,000. Several lines of evidence indicate that this polypeptide is responsible for carbonic anhydrase activity: (a) it appears following the transfer of C. reinhardtii from growth on 5% CO2 to growth on air levels of CO2, (b) it is located in the periplasmic space or associated with the cell wall, like the bulk of the carbonic anhydrase activity, (c) it binds dansylamide, an inhibitor of the enzyme which fluoresces upon illumination with ultraviolet light, (d) antibodies which inhibit carbonic anhydrase activity only cross-react with this 37,000 dalton species. 相似文献
9.
Investigations using steady-state culture conditions indicate that carbonic anhydrase activity is correlated to the photosynthetic rate in Euglena in some but not all circumstances. When cultures grown with 5% CO2 were changed to air growth, the photosynthetic rate was independent of the carbonic anhydrase activity. While experiments using the inhibitor acetazolamide indicated a close correlation between photosynthetic capacity and carbonic anhydrase activity, the inhibitor was found to be nonspecific. Acetazolamide altered photosystem activities directly as measured by the photoreduction of DCPIP in chloroplast preparations, whole-cell fluorescence transients of chlorophyll a, and by whole chain photoelectron flow. Ethoxzolamide, another inhibitor of carbonic anhydrase, was also found to inhibit photosystem activities, i.e., the photoreduction of DCPIP, and in vivo photoelectron flow, at high concentrations. Cells grown in 5% CO2 were less sensitive to the effects of acetazolamide than cells exposed to air. The rate of electron flow in chloroplasts from cells grown with 5% CO2 and exposed to 10 mM acetazolamide was 2.5-fold faster than that of chloroplasts from air-grown cells exposed to the same concentration of inhibitor. The whole cell chlorophyll a fluorescence transients of cultures grown with high CO2 were completely different from those of air-grown cells and also showed fewer effects on exposure to acetazolamide. These results suggest a reevaluation of the hypothesis that carbonic anhydrase activity regulates photosynthesis. It is also apparent that results from air-grown and 5% CO2-grown cultures cannot be directly compared in such studies. 相似文献
10.
Carbonic anhydrase activity in a calcium-mobilizing epithelium of the crustacean Orchestia cavimana during molting 总被引:1,自引:0,他引:1
Summary We investigated the involvement of the enzyme, carbonic anhydrase, in the calcification-decalcification processes occurring in the posterior caeca of the midgut of the terrestrial crustacean, Orchestia cavimana, before and after exuviation. This enzyme was ultrahistochemically localized throughout the membranes of the caecal epithelium as well as extracellularly, i.e., within pre-exuvial calcareous concretions and postexuvial calcified spherules. During the molt cycle, the pattern of carbonic anhydrase activity in the posterior caeca was correlated with the calcium content at this level. Acetazolamide treatment in vivo inhibited about 50% of the calcium uptake during both pre-exuvial secretion and postexuvial reabsorption. The role of carbonic anhydrase in this mineralizing-demineralizing epithelium is discussed and compared with that of other mechanisms involved in this calcium turnover. 相似文献
11.
It was previously reported that magnesium ion inhibited carbonic anhydrase (Bamberger and Avron 1975 Plant Physiol 56: 481-485). Studies with partially purified carbonic anhydrase from spinach (Spinacia oleracea L.) chloroplasts show that the effect was the result of the chloride counterion and not the magnesium ion. Enzyme activity was reduced 50% upon addition of 3 to 10 millimolar MgCl2 or KCl while all additions of MgSO4 between 0.3 and 10 millimolar were mildly stimulatory. 相似文献
12.
Comparative studies on acetazolamide teratogenesis in pregnant rats, rabbits, and rhesus monkeys 总被引:1,自引:0,他引:1
Acetazolamide produces a characteristic forelimb reduction deformity when administered to pregnant rodents. Past studies indicated that non-rodent species (rabbit and monkey) are resistant to this effect. The present studies confirmed this fact and demonstrated that transport of acetazolamide into the rabbit embryo was similar to that in sensitive rat embryos. In monkeys, however, the concentrations of acetazolamide within maternal plasma and embryo were much lower than in rats. Carbonic anhydrase activity was also measured since inhibition of this enzyme is the primary pharmacologic effect of acetazolamide. Again the rabbit embryo had carbonic anhydrase specific activity levels similar to that of the rat. Monkey embryos, on the other hand, contained negligible levels of enzyme activity during the presumed sensitive period of development. Thus the resistance of monkey embryos to acetazolamide teratogenesis may be due to low carbonic anhydrase activity and/or the small amount of drug reaching the embryo. No basis for the resistance of rabbit embryos to acetazolamide teratogenesis was uncovered. 相似文献
13.
At concentrations of 100–200 M, ethoxyzolamide, a lipophilic inhibitor of carbonic anhydrase, considerably (by 60%) inhibited light-induced CO2-dependent oxygen evolution in pea protoplasts at the optimum concentration of inorganic carbon (100 M CO2) in the medium. At the same concentrations of the inhibitor, electron transport in isolated pea thylakoids was inhibited only by 6–9%. Acetazolamide, a water-soluble inhibitor of carbonic anhydrase, affected neither the rate of CO2-dependent O2evolution in protoplasts nor electron transport in thylakoid membranes. A light-dependent proton uptake by protoplasts was demonstrated. At pH 7.2, the induction kinetics and the rate of proton uptake were similar to those for CO2-dependent O2evolution. The rate of proton uptake was decreased twofold by 1 mM acetazolamide. This fact agrees with the notion that a membrane-bound carbonic anhydrase is operative in the plasma membrane of higher plant cells. A mechanism of its functioning is suggested. Possible functions of carbonic anhydrases in the cells of C3-plants are discussed. 相似文献
14.
Carbonic Anhydrase-Dependent Inorganic Carbon Uptake by the Red Macroalga, Chondrus crispus 总被引:2,自引:1,他引:1
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The rate of photosynthetic carbon uptake of Chondrus crispus Stack-house plants, at various CO2 concentrations and pretreated with carbonic anhydrase (CA) inhibitors, was determined using an air-suspension, differential infra-red gas analyzer technique. It was found that the CA inhibitors, acetazolamide, dextran-bound acetazolamide (DBI, which does not permeate cell membranes), and subtilisin (a protease that attacks the cell surface) inhibit photosynthetic carbon uptake in C. crispus. Inhibition was greatest at low CO2 concentrations, and decreased at CO2 saturation. Acetazolamide inhibited carbon uptake to a greater extent than DBI. The data support the conclusion that C. crispus plants utilize HCO3− for photosynthesis, and that both cell-surface and internal CA are involved in the photosynthetic uptake of inorganic carbon. 相似文献
15.
Francesca Viparelli Simona Maria Monti Giuseppina De Simone Alessio Innocenti Andrea Scozzafava Yan Xu François M.M. Morel Claudiu T. Supuran 《Bioorganic & medicinal chemistry letters》2010,20(16):4745-4748
We investigated the catalytic activity and inhibition of both the zinc and cadmium-containing R1 fragment of the ζ-class carbonic anhydrase (CA, EC 4.2.1.1) from the marine diatom Thalassiosira weissflogii. Our data prove that these enzymes are not only very efficient catalysts for the physiological reaction, but also sensitive to sulfonamide and anion inhibitors, with inhibition constants from the nanomolar to millimolar range. Acetazolamide inhibited the two enzymes with KIs in the range of 58–92 nM. The best anion inhibitors of Cd-R1 were thiocyanate, sulfamate and sulfamide, with KIs of 10–89 μM, whereas the best Zn-R1 anion inhibitors were sulfamate and sulfamide with KIs of 60–72 μM. These enzymes were only weakly inhibited by chloride, bromide or sulfate, main anion components of sea water, with inhibition constants in the range of 0.24–0.85 mM. Thus, similarly to CAs belonging to other classes, the ζ-class CA (with either cadmium or zinc ions at the active site) was inhibited by both anions and sulfonamides. 相似文献
16.
Evidence That an Internal Carbonic Anhydrase Is Present in 5% CO(2)-Grown and Air-Grown Chlamydomonas 总被引:5,自引:5,他引:0
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Inorganic carbon (Ci) uptake was measured in wild-type cells of Chlamydomonas reinhardtii, and in cia-3, a mutant strain of C. reinhardtii that cannot grow with air levels of CO2. Both air-grown cells, that have a CO2 concentrating system, and 5% CO2-grown cells that do not have this system, were used. When the external pH was 5.1 or 7.3, air-grown, wild-type cells accumulated inorganic carbon (Ci) and this accumulation was enhanced when the permeant carbonic anhydrase inhibitor, ethoxyzolamide, was added. When the external pH was 5.1, 5% CO2-grown cells also accumulated some Ci, although not as much as air-grown cells and this accumulation was stimulated by the addition of ethoxyzolamide. At the same time, ethoxyzolamide inhibited CO2 fixation by high CO2-grown, wild-type cells at both pH 5.1 and 7.3. These observations imply that 5% CO2-grown, wild-type cells, have a physiologically important internal carbonic anhydrase, although the major carbonic anhydrase located in the periplasmic space is only present in air-grown cells. Inorganic carbon uptake by cia-3 cells supported this conclusion. This mutant strain, which is thought to lack an internal carbonic anhydrase, was unaffected by ethoxyzolamide at pH 5.1. Other physiological characteristics of cia-3 resemble those of wild-type cells that have been treated with ethoxyzolamide. It is concluded that an internal carbonic anhydrase is under different regulatory control than the periplasmic carbonic anhydrase. 相似文献
17.
M K Koester A M Register E A Noltmann 《Biochemical and biophysical research communications》1977,76(1):196-204
Rabbit muscle cytosol extract contains a basic protein which represents about 2% of the total cytosol protein. It contains zinc in a 1:1 stoichiometric ratio, based on a molecular weight of 30,000, and it catalyzes the hydration of CO2. It is immunochemically distinct from the high and low activity forms of rabbit blood carbonic anhydrase. It has comparatively poor activity as an esterase, and about 20% of the CO2 hydratase activity of the rabbit blood low activity carbonic anhydrase. This CO2 hydratase activity is not inhibited by acetazolamide at concentrations which totally inhibit the activity of the blood carbonic anhydrases. The evidence obtained to date, though circumstantial, suggests that this basic metalloprotein is a carbonic anhydrase derived from a third genetic locus with properties considerably different from those of the mammalian carbonic anhydrases heretofore identified. 相似文献
18.
Supernatant obtained after high-speed centrifugation of disrupted thylakoids that had been washed free from extrathylakoid carbonic anhydrases demonstrated carbonic anhydrase activity that was inhibited by the specific inhibitors acetazolamide and ethoxyzolamide. A distinctive feature of the effect of Triton X-100 on this activity also suggested that the source of the activity is a soluble protein. Native electrophoresis of a preparation obtained using chromatography with agarose/mafenide as an affinity sorbent revealed one protein band with carbonic anhydrase activity. The same protein was revealed in a mutant deficient in soluble stromal carbonic anhydrase β-CA1, and this indicated that the newly revealed carbonic anhydrase is not a product of the At3g01500 gene. These data imply the presence of soluble carbonic anhydrase in the thylakoid lumen of higher plants. 相似文献
19.
Microbial carbonic anhydrase promotes carbonate deposition, which is important in the formation and evolution of global carbon cycle and geological processes. A kind of bacteria producing extracellular carbonic anhydrase was selected to study the effects of temperature, pH value and Ca2+ concentration on bacterial growth, carbonic anhydrase activity and calcification rate in this paper. The results showed that the activity of carbonic anhydrase at 30 °C was the highest, which was beneficial to the calcification reaction, calcification rate of CaCO3 was the fastest in alkaline environment with the initial pH value of 9.0. When the Ca2+ concentration was 60 mM, compared with other Ca2+ concentration, CA bacteria could grow and reproduce best, and the activity of bacteria was the highest, too low Ca2+ concentration would affect the generation of CaCO3, while too high Ca2+ concentration would seriously affect the growth of bacteria and reduce the calcification rate. Finally, the mechanism of CaCO3 precipitation induced by microbial carbonic anhydrase was studied. Carbonic anhydrase can accelerate the hydration of CO2 into HCO3−, and react with OH− and Ca2+ to form CaCO3 precipitation in alkaline environment and in the presence of calcium source. 相似文献
20.
Süleyman Yılmaz Yusuf Akbaba Bünyamin Özgeriş Leyla Polat Köse 《Journal of enzyme inhibition and medicinal chemistry》2016,31(6):1484-1491
A series of carbamate derivatives were synthesized and their carbonic anhydrase I and II isoenzymes and acetylcholinesterase enzyme (AChE) inhibitory effects were investigated. All carbamates were synthesized from the corresponding carboxylic acids via the Curtius reactions of the acids with diphenyl phosphoryl azide followed by addition of benzyl alcohol. The carbamates were determined to be very good inhibitors against for AChE and hCA I, and II isoenzymes. AChE inhibition was determined in the range 0.209–0.291?nM. On the other hand, tacrine, which is used in the treatment of Alzheimer’s disease possessed lower inhibition effect (Ki: 0.398?nM). Also, hCA I and II isoenzymes were effectively inhibited by the carbamates, with inhibition constants (Ki) in the range of 4.49–5.61?nM for hCA I, and 4.94–7.66?nM for hCA II, respectively. Acetazolamide, which was clinically used carbonic anhydrase (CA) inhibitor demonstrated Ki values of 281.33?nM for hCA I and 9.07?nM for hCA II. The results clearly showed that AChE and both CA isoenzymes were effectively inhibited by carbamates at the low nanomolar levels. 相似文献